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Cholesterol metabolism in pancreatic cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

New Pancreatic Treatment Shows Promise

http://www.genengnews.com/gen-news-highlights/new-pancreatic-treatment-shows-promise/81252686/

Study demonstrates how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis. [NIH].   http://www.genengnews.com/Media/images/GENHighlight/thumb_May4_2016_NIH_PancreaticCancerCells8616346835.jpg

Scientists say they have shown how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis, pointing to a potential new treatment using drugs previously developed for atherosclerosis.

“We show for the first time that if you control the cholesterol metabolism you could reduce pancreatic cancer spread to other organs,” said Ji-Xin Cheng, Ph.D., a professor in Purdue University’s Weldon School of Biomedical Engineering and Department of Chemistry. “We chose pancreatic cancer to test this approach because it is the most aggressive disease of all the cancers.”

Dr. Cheng had previously led a team of researchers discovering a link between prostate cancer’s aggressiveness and the accumulation of a compound produced when cholesterol is metabolized in cells, findings that could bring new diagnostic and treatment methods. The new study involved researchers at the Purdue Center for Cancer Research and School of Biomedical Engineering, the Indiana University Simon Cancer Center and School of Medicine, and Purdue’s Department of Biological Sciences, Department of Comparative Pathobiology, and Department of Biochemistry.

The findings, detailed in a paper (“Abrogating Cholesterol Esterification Suppresses Growth and Metastasis of Pancreatic Cancer”) just published in Oncogene, suggest that a class of drugs previously developed to treat atherosclerosis could be repurposed for treatment of pancreatic cancer and other forms of cancer. Atherosclerosis is the buildup of fats, cholesterol, and other substances in arteries, restricting blood flow.

The researchers found accumulations of the compound cholesteryl ester in human pancreatic cancer specimens and cell lines, demonstrating a link between cholesterol esterification and metastasis. Excess quantities of cholesterol result in cholesteryl ester being stored in lipid droplets within cancer cells.

“The results of this study demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification,” said Jingwu Xie, Ph.D., the Jonathan and Jennifer Simmons Professor at the Indiana University School of Medicine and a researcher at the Indiana University Melvin and Bren Simon Cancer Center.

The paper’s lead author is Purdue postdoctoral fellow Junjie Li, Ph.D. Purdue researchers have developed an analytical tool, Raman spectromicroscopy, that allows compositional analysis of single lipid droplets in living cells.

“We identified an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines,” Dr. Li said. “Depletion of cholesterol esterification significantly reduced pancreatic tumor growth and metastasis in mice.”

Findings show that drugs like avasimibe, previously developed for treatment of atherosclerosis, reduced the accumulation of cholesteryl ester. Pancreatic cancer usually kills within a few months of diagnosis. It is hoped the potential new treatment might extend life of these patients for a year, Cheng said.

The accumulation of cholesteryl ester is controlled by an enzyme called acyl-coenzyme A acyltransferase-1 (ACAT-1), and findings have correlated a higher expression of the enzyme with a poor survival rate for patients. The researchers analyzed tissue samples from pancreatic cancer patients and then tested the drug treatment in a type of laboratory mice referred to as an orthotopic mouse model, developed at the IU School of Medicine. Specimens of human pancreatic tissues were obtained from the Simon Cancer Center Solid Tissue Bank.

Imaging showed a decrease of the number of lipid droplets, and Raman spectral analysis verified a significant reduction of cholesteryl ester in the lipid droplets, suggesting that avasimibe acted by blocking cholesterol esterification. The drug did not induce weight loss, and there was no apparent organ toxicity in the liver, kidney, lung and spleen, Dr. Cheng said.

Findings also showed that blocking storage of cholesteryl ester causes cancer cells to die, specifically due to damage to the endoplasmic reticulum, a workhorse of protein and lipid synthesis.

“By using avasimibe, a potent inhibitor of ACAT-1, we found that pancreatic cancer cells were much more sensitive to ACAT-1 inhibition than normal cells,” added Dr. Cheng.

Additional research confirmed that the anticancer effect of avasimibe is specific to ACAT-1 inhibition. The researchers performed various biochemical assays and “genetic ablation” to confirm the drug’s anticancer effect.

“The results showed that avasimibe treatment for four weeks remarkably suppressed tumor size and largely reduced tumor growth rate,” said paper co-author Timothy Ratliff, the Robert Wallace Miller Director of Purdue’s Center for Cancer Research. “Metastatic lesions in lymph nodes and distant organs also were assessed at the end of the study. A much higher number of metastatic lesions in lymph nodes were detected in the control group than the avasimibe-treated group.”

Each mouse in the control group showed at least one metastatic lesion in the liver. In contrast, only three mice in the avasimibe-treated group showed single lesion in liver.

 

Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer

J Li1, D Gu2, S S-Y Lee1, B Song1, S Bandyopadhyay3, S Chen4, S F Konieczny3,5, T L Ratliff5,6, X Liu5,7, J Xie2 and J-X Cheng1,5
O
ncogene 2 May 2016;                                         http://dx.doi.org:/10.1038/onc.2016.168

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Metastasis is the major cause of cancer-related mortality. Though localized tumors can often be treated by surgery or other therapies, treatment options for metastatic diseases are limited. Cancer metastasis has been revealed to be a multiple step process, including cancer cell migration, local invasion, intravasation, circulation through blood and lymph vessels, extravasation, survival and colonization in distant organs.1, 2, 3Mediators identified in these processes have provided the basis for the development of therapies to target metastasis. Current therapeutic strategies for treating metastatic tumors mainly focus on targeting the adhesive molecules and extracellular proteases.4However, these therapeutics have not been proven to be effective in clinical trials, partially owing to the various escape mechanisms used by the metastatic cancer cells.2, 5, 6 Thus, an unmet need exists to develop new therapeutic strategies for treating metastatic cancers.

Recent advances in cancer metabolism have unveiled many potential therapeutic targets for cancer treatment. Metabolic reprogramming, a strategy used by cancer cells to adapt to the rapid proliferation, is being recognized as a new hallmark of cancer.7 Substantial studies have found increased glycolysis, glutaminolysis, nucleotide and lipid synthesis in cancer cells.7, 8, 9,10 Considering that altered metabolic pathways only happen in cancer cells but not in normal cells, targeting these pathways may provide cancer-specific treatments. A number of inhibitors of metabolic enzymes, such as glycolysis inhibitors, are under clinical trials as targeted cancer therapeutics.11

Of various metabolic pathways, lipid metabolism has been suggested to have an important role in cancer cell migration, invasion and metastasis.12 A recent study reported that surrounding adipocytes provide energy source for ovarian cancer cells to promote its rapid growth and metastasis.13 Blocking lipidde novo synthesis pathway has been shown to suppress tumor regrowth and metastasis after anti-angiogenesis treatment withdrawal.14 In parallel, lipolysis by the enzyme monoacylglycerol lipase was shown to regulate the fatty acid network, which promotes cancer cell migration, invasion and growth.15

Cholesterol, a critical component of the plasma membrane, is also implied to be correlated to cancer metastasis.16 It has been shown that prostate cancer bone metastases contain a high level of cholesterol.17 Modulation of cholesterol level in plasma membrane was shown to regulate the capability of cell migration.18, 19Moreover, cholesterol-enriched lipid rafts were shown to have an essential role in cancer cell adhesion and migration.20 Mammalian cells obtain cholesterol either from de novo synthesis or from the uptake of low-density lipoprotein (LDL).21 Inside cells, excess free cholesterol is esterified and stored as cholesteryl ester (CE) in lipid droplets (LDs), which is mediated by acyl-CoA cholesterol acyltransferase (ACAT).22 Increased CE level has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26Despite these advances, the role of cholesterol esterification in cancer progression, especially in cancer metastasis, is not well understood.

In this article, we report a link between cholesterol esterification and metastasis in pancreatic cancer. Using stimulated Raman scattering (SRS) microscopy and Raman spectroscopy to map LDs stored inside single cells and analyze the composition of individual LDs, we identified an aberrant accumulation of CE in human pancreatic cancer specimens and cell lines. Abrogation of cholesterol esterification, either by inhibiting ACAT-1 enzyme activity or by shRNA knockdown of ACAT-1 expression, significantly reduced pancreatic tumor growth and metastasis in an orthotopic mouse model. Mechanistically, inhibition of cholesterol esterification disturbed cholesterol homeostasis by increasing intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum (ER) stress and eventually led to apoptosis.

In this study, we revealed a link between CE accumulation and pancreatic cancer metastasis. Accumulation of CE via ACAT-1 provides a mechanism to keep high metabolic activity and avoid toxicity from excess free cholesterol. Previously, CE has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26 Inhibition of cholesterol esterification was shown to suppress tumor growth or cancer cell proliferation.24, 25, 26 Here, we demonstrate that inhibition of cholesterol esterification can be used to treat metastatic pancreatic cancer.

Cholesterol is an essential lipid having important roles in membrane construction, hormone production and signaling.21Aberrant cholesterol metabolism is known to be associated with cardiovascular diseases and cancers.35, 36 Statins, inhibitors of HMG-CoA reductase, have been explored as potential therapies for pancreatic cancer.37 However, statins were not associated with a reduced risk of pancreatic cancer in clinical trials.38 One possible reason is that HMG-CoA reductase is also required for downstream protein prenylation, a critical process for protein activation.39Thus, the effect of statin is not just inhibiting cholesterol synthesis, but also other pathways which may render toxicity to normal cells. This non-specific toxicity is a possible reason for the limited anti-cancer outcome of statin in clinical trials.

Our study identified cholesterol esterification as a novel target for suppression of pancreatic cancer proliferation and metastasis. Inhibitors of ACAT-1 are expected to have great value as cancer-targeting therapeutics, as CE accumulation only occurs in cancer tissues or cell lines. Our animal studies with avasimibe treatment showed no adverse effect to the animals at a dosage of 15mg/kg. More importantly, modulation of cholesterol esterification suppressed not only tumor growth but also tumor metastasis. These results are expected to stimulate further biological studies to fully appreciate the role of cholesterol metabolism in cancer initiation and progression. As CE accumulation happens in several types of aggressive cancer, blocking cholesterol esterification could be pursued as a therapeutic strategy for other types of cancers. By combining with existing chemotherapies, such as gemcitabine, we believe this metabolic treatment possesses high possibilities to extend patients’ survival time by retarding cancer progression and metastasis.

The molecular mechanism that links CE accumulation to cancer aggressiveness needs further studies. One possible mechanism is that cholesterol esterification keeps signaling pathways active by maintaining a low free cholesterol environment. One of the possible targets is the caveolin-1 signaling pathway. Caveolin-1, a regulator of cellular cholesterol homeostasis, is considered as a marker for pancreatic cancer progression.11 Particularly, a promoting role of caveolin-1 in pancreatic cancer metastasis has been reported.40 Our preliminary studies showed ACAT-1 inhibition reduced the expression level of SREBP1, caveolin-1 and phosphorylated ERK1/2 (unpublished data). The effect on caveolin-1 is probably mediated by SREBP1, which senses the intracellular cholesterol homeostasis.41 Meanwhile, caveolin-1 may have an important role in mediating the action of SREBP1 on MAPK pathways,42, 43 which are known to have essential roles in cancer cell metastasis.44 Therefore, it is possible that increased free cholesterol level induced by ACAT-1 inhibition inactivates SREBP1, leading to downregulation of caveolin-1/MAPK pathway, which contributes to the reduced cancer aggressiveness.

Besides the caveolin-1/MAPK signaling, other possibilities include the potential alteration of the membrane composition, such as lipid rafts, by ACAT-1 inhibition. Lipid rafts are known to provide platforms for multiple cellular signaling pathways.20 Thus, modulation of cholesterol metabolism is likely to have more profound effects via other signaling pathways. Future studies are needed to fully elucidate the molecular mechanism.

 

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Role of Inflammation in Disease

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Inflamed  

The debate over the latest cure-all craze.

BY

Medical Dispatch NOVEMBER 30, 2015 ISSUE     http://www.newyorker.com/magazine/2015/11/30/inflamed

 

The National Institutes of Health recently designated inflammation a priority.

 

The National Institutes of Health recently designated inflammation a priority.
CREDIT ILLUSTRATION BY CHAD HAGEN

 

Several years ago, I fell at the gym and ripped two tendons in my wrist. The pain was excruciating, and within minutes my hand had swollen grotesquely and become hot to the touch. I was reminded of a patient I’d seen early in medical school, whose bacterial infection extended from his knee to his toes. Latin was long absent from the teaching curriculum, but, as my instructor examined the leg, he cited the four classic symptoms of inflammation articulated by the Roman medical writer Celsus in the first century: rubor, redness; tumor, swelling; calor, heat; and dolor, pain. In Latin, inflammatio means “setting on fire,” and as I considered the searing pain in my injured hand I understood how the condition earned its name.

Inflammation occurs when the body rallies to defend itself against invading microbes or to heal damaged tissue. The walls of the capillaries dilate and grow more porous, enabling white blood cells to flood the damaged site. As blood flows in and fluid leaks out, the region swells, which can put pressure on surrounding nerves, causing pain; inflammatory molecules may also activate pain fibres. The heat most likely results from the increase in blood flow.
The key white blood cell in inflammation is called a macrophage, and for decades it has been a subject of study in my hematology laboratory and in many others. Macrophages were once cast as humble handmaidens of the immune system, responsible for recognizing microbes or debris and gobbling them up. But in recent years researchers have come to understand that macrophages are able to assemble, within themselves, specialized platforms that pump out the dozens of molecules that promote inflammation. These platforms, called inflammasomes, are like pop-up factories—quickly assembled when needed and quickly dismantled when the crisis has passed.

For centuries, scientists have debated whether inflammation is good or bad for us. Now we believe that it’s both: too little, and microbes fester and spread in the body, or wounds fail to heal; too much, and nearby healthy tissue can be degraded or destroyed. The fire of inflammation must be tightly controlled—turned on at the right moment and, just as critically, turned off. Lately, however, several lines of research have revealed that low-level inflammation can simmer quietly in the body, in the absence of overt trauma or infection, with profound implications for our health. Using advanced technologies, scientists have discovered that heart attacks, diabetes, and Alzheimer’s disease may be linked to smoldering inflammation, and some researchers have even speculated about its role in psychiatric conditions.

As a result, understanding and controlling inflammation has become a central goal of modern medical investigation. The internal research arm of the National Institutes of Health recently designated inflammation a priority, mobilizing several hundred scientists and hundreds of millions of dollars to better define its role in health and disease; in 2013, the journal Science devoted an entire issue to the subject. This explosion in activity has captured the public imagination. In best-selling books and on television and radio talk shows, threads of research are woven into cure-all tales in which inflammation is responsible for nearly every malady, and its defeat is the secret to health and longevity. New diets will counter the inflammation simmering in your gut and restore your mental equilibrium. Anti-inflammatory supplements will lift your depression and ameliorate autism. Certain drugs will tamp down the silent inflammation that degrades your tissues, improving your health and extending your life. Everything, and everyone, is inflamed.

Such claims aside, there is genuine evidence that inflammation plays a role in certain health conditions. In atherosclerosis, blood flow to the heart or the brain is blocked, resulting in a heart attack or a stroke. For a long time, atherosclerosis was thought to result mainly from eating fatty foods, which clogged the arteries. “Atherosclerosis was all about fats and grease,” Peter Libby, a professor at Harvard Medical School and a cardiologist at Brigham and Women’s Hospital, in Boston, told me recently. “Most physicians saw atherosclerosis as a straight plumbing problem.”

During his cardiology training, Libby studied immunology, and he became fascinated with the work of Rudolf Virchow, a nineteenth-century German pathologist. Virchow speculated that atherosclerosis might be an active process, caused by inflamed blood vessels, not one caused simply by the accumulation of fat. In the mid-nineteen-nineties, in studies with mice, Libby, working in parallel with other groups of scientists, found that low-density lipoproteins—LDLs, those particles of “bad” cholesterol—can work their way into the lining of arteries. There, they sometimes trigger an inflammatory response, which can cause blood clots that block the artery. Libby and others began to understand that atherosclerosis wasn’t a mere plumbing problem but also an immune disease—“our body’s defenses turned against ourselves,” he told me.

Paul Ridker, a cardiovascular expert and a colleague of Libby’s at Harvard and Brigham and Women’s, moved the research beyond the laboratory. He found that many patients who’d had heart attacks, despite lacking factors such as high blood pressure, high cholesterol, and a history of smoking, had an elevated level of C-reactive protein, a molecule produced in response to inflammation, in their blood. After demonstrating, in a separate study, that cholesterol-reducing statins could also reduce C-reactive-protein levels, Ridker launched the Jupiter trial, in which people with elevated levels of C-reactive protein but normal cholesterol levels were given a placebo or a statin medication. In 2008, the published results showed that the subjects who received the statin saw their levels of C-reactive protein drop and were less likely three and a half years later to suffer a heart attack. This suggested that elevated cholesterol isn’t the only factor at work in cardiovascular disease, and that in some cases statins, acting as anti-inflammatory agents, could be used to treat the condition.
The benefit was modest: the statin treatment reduced the risk of heart attack in only about one per cent of the patients. Still, that figure is statistically significant, and for one in every hundred patients—a hundred in every ten thousand—it’s meaningful. An independent safety-monitoring board ended the study early, saying that it was unethical to continue once it was clear that statins provided a benefit not available to the subjects on the placebo. (Critics argue that shortening the trial, which was funded by a drug company, exaggerated the potential benefits and underestimated long-term harm, but the researchers strongly disagree.) The N.I.H. and other scientific groups are funding new studies to further explore whether anti-inflammatory drugs—for example, low doses of immunomodulatory agents that are used for treating severe arthritis—can help prevent cardiovascular disease.
Another chronic condition that has been linked to inflammation is Type II diabetes. People with this condition can’t adequately use insulin, a molecule that enables the body’s cells to take glucose out of the bloodstream and derive energy from it. Their organs fail and glucose builds to dangerous levels in the blood. Recently, researchers have found macrophages in the pancreases of people with Type II diabetes. The macrophages release inflammatory molecules that are thought to impair insulin activity. One of these inflammatory molecules is called interleukin-1, and in 2007 the New England Journal of Medicine reported on a clinical trial in which an interleukin-1 blocker proved to be modestly effective at lowering blood-sugar levels in Type II diabetics. This suggests that, by blocking inflammation, it might be possible to restore insulin activity and alleviate some of the symptoms of diabetes.

Alzheimer’s disease, too, seems to show a link to inflammation. Alzheimer’s results from the buildup of amyloid and tau proteins in the brain; specialized cells called glial cells, which are related to macrophages, recognize these proteins as debris and release inflammatory molecules to get rid of them. This inflammation is thought to further impair the working of neurons, worsening Alzheimer’s. The connection is tantalizing, but it’s important to note that it doesn’t mean that inflammation causes Alzheimer’s. Nor is there strong evidence that inflammation contributes to other forms of dementia where the brain isn’t filled with protein debris. And in clinical trials anti-inflammatory drugs like naproxen and ibuprofen have failed to ameliorate or prevent Alzheimer’s.

 

On September 18, 2015, scientists at the N.I.H. convened a meeting to publicly present their research priorities, one of which is to decipher the consequences of inflammation. It’s increasingly apparent that inflammation plays some role in many health conditions, but scientists are far from grasping the nature of that relationship, the mechanisms involved, or the extent to which treating inflammation is helpful.

“We really don’t know how much inflammation contributes to diabetes, Alzheimer’s, depression, and other disorders,” Michael Gottesman, a director of research at the N.I.H., told me. “We know a lot about the mouse and its immune response. Much, much less is understood in humans. As we learn more, we see how much more we need to learn.” Gottesman pointed out that, of the thousand or so proteins circulating in our bloodstream, about a third are involved in inflammation and in our immune response, so simply detecting their presence doesn’t reveal much about their potential involvement in any particular disease. “Correlation is not causation,” he emphasized. “Because you find an inflammatory protein in a certain disorder, it doesn’t mean that it is causing that disorder.”
This lack of certainty hasn’t dampened the enthusiasm of a growing number of doctors who believe that inflammation is the source of a wide range of conditions, including dementia, depression, autism, A.D.H.D., and even aging. One of the most prominent such voices is that of Mark Hyman, whose books—including “The Blood Sugar Solution 10-Day Detox Diet”—are best-sellers. Hyman serves as a personal health adviser to Bill and Hillary Clinton and to the King and Queen of Jordan. Recently, he was recruited by the Cleveland Clinic with millions of dollars in funding to establish a center based on his ideas. Trained in family medicine, Hyman told me that he considers himself a new type of doctor. “I am a doctor who treats root causes and addresses the body as a dynamic system,” he wrote in an e-mail. “Being an inflammalogist is part of that.”

Studies with human subjects clearly indicate that, in cases where inflammation underlies a chronic condition, the inflammation is local: in the arteries (heart disease); or in the brain (Alzheimer’s); or in the pancreas (diabetes). And though there are associations between various forms of inflammatory disease—for example, people with psoriasis or periodontal disease have a somewhat higher risk of heart disease—it has not been proved that there is a causal connection. Hyman and other doctors, such as the neurologist David Perlmutter, promote a more radical idea: that certain foods and environmental toxins cause smoldering inflammation, which somehow spreads to other areas of the body, including the brain, degrading one’s health, mental acuity, and life span.

The notion of a gut-brain connection seems to derive from studies with mice, including one that showed that introducing a bacterium into a mouse’s gastrointestinal tract led to behavioral changes, such as a reluctance to navigate mazes. But there’s scant evidence that anything similar happens in people, or any rigorous study to show that “anti-inflammatory diets” reduce depression. Earlier this year, the journal Brain, Behavior, and Immunity published a meta-analysis of more than fifty clinical studies that found inflammatory molecules in patients with depression. The paper revealed that there was little consistency from study to study about which molecules correlated to the condition. Steven Hyman, a former director of the National Institute of Mental Health and now the head of the Stanley Center at the Broad Institute (and no relation to Mark Hyman), in Cambridge, Massachusetts, noted that depression is “one of those topics where exuberant theorization vastly outstrips the data.”

Nonetheless, Mark Hyman holds fast to his view. “Inflammation is the final common pathway for pretty much all chronic diseases,” he told me. His recommended solution is an “anti-inflammatory diet”—omitting sugar, caffeine, beans, dairy, gluten, and processed foods, as well as taking a variety of supplements, including probiotics, fish oil, Vitamins C and D, and curcumin, a key molecule in turmeric. Hyman introduced me to one of the patients he had treated with his anti-inflammatory diet and supplements, a forty-seven-year-old hedge-fund manager in Cambridge named Jim Silverman. Two decades ago, Silverman began noticing blood in his stool. A colonoscopy resulted in a diagnosis of ulcerative colitis. In the ensuing years, Silverman was treated by gastroenterologists with aspirin-based medication, anti-inflammatory suppositories, and even corticosteroids, but the problem persisted. Then, five years ago, on a flight home from a business conference, he happened to sit next to Hyman, who told him that he could cure colitis.
“I thought, What a bullshitter,” Silverman said. He travelled anyway to Hyman’s UltraWellness Center, in Lenox, Massachusetts, to consult with him. Hyman told him that dairy was inflaming his bowel. Silverman was skeptical, but he kept track of his diet and bleeding episodes, and ultimately concluded that restricting dairy products resulted in long periods without bleeding. He now thinks that he could be suffering from a dairy allergy. In addition to avoiding dairy products, he continues to follow the anti-inflammatory regimen of supplements prescribed by Hyman. “I’m just taking it because I’m doing well,” he said. “I have no idea if it’s doing anything, but I don’t want to rock the boat.”

I asked Gary Wu, a professor of gastroenterology at the Perelman School of Medicine, at the University of Pennsylvania, and one of the world’s experts on the gut microbiome, about the alleged value of treating inflammatory bowel disease by restricting specific foods. Recently, in the journal Gastroenterology, Wu and his colleagues published a comprehensive review of scientific studies on diet and inflammatory bowel disease. They found only two dietary interventions that had been proved to reduce inflammation: an “elemental diet,” which is a liquid mixture of amino acids, simple sugars, and triglycerides, and a slightly more complex liquid diet. The liquid mixtures are typically administered with a tube placed through the nose. “The diet is not palatable,” Wu said. “And you don’t eat during the day. There is no intake of whole foods at all.”

David Agus, a cancer specialist and a professor of medicine and engineering at the University of Southern California, is equally skeptical of Hyman’s claims for the anti-inflammation diet. Agus, who is perhaps best known for being the doctor on “CBS This Morning,” recently received a multimillion-dollar grant from the National Cancer Institute to study how inflammation may spur the growth of tumors. “This notion that foods cause inflammation and foods can block inflammation, there’s zero data that it changes clinical outcomes,” he told me. “If the idea gets people to eat fruits and vegetables, I love it, but it’s not real.” Agus noted that vitamins don’t counter inflammation, and that it’s been shown, in rigorous clinical trials, that they may increase one’s risk of developing cancer.
Still, Agus views inflammation as a component not only of cancer but also of chronic diseases like diabetes and dementia. Rather than special diets, he supports preventively taking approved anti-inflammatory medications, such as aspirin and statins, and scrupulously scheduling the standard vaccinations in order to prevent infections. In “The End of Illness,” Agus encourages the reader to “reduce your daily dose of inflammation” by, among other things, not wearing high heels, since these can inflame your feet and the inflammation could possibly affect your vital organs. When I pressed him on that suggestion, he told me, “What I meant is that if your feet hurt all day it’s probably not a good thing. The downside is you just wear a different pair of shoes. The upside is it gave you an understanding of inflammation and its role in disease.”

Mark Hyman, at times, acknowledges the possible limits of his paradigm. When I asked him about the alleged link among gut inflammation, diet, and psychological disorders, he conceded that some of his evidence was anecdotal, derived from his own clinical practice. He mentioned the case of a child with asthma, eczema, and A.D.H.D., whom he treated with “an elimination diet, taking him off processed foods, and giving him supplements.” The child’s allergic problems improved and his behavior was markedly better, Hyman said: “It was a light-bulb moment. I saw secondary effects on the brain that came out of treating physical problems.”

He also cited studies of patients with rheumatoid arthritis, a painful and debilitating auto-immune condition that inflames and erodes the joints, who became less depressed after being treated with inflammatory blockers. But had the anti-inflammatory treatment directly lifted their depression, or had their mood improved simply because they were more mobile and in less pain? I told Hyman that it was hard to connect the dots. “For sure,” he said.

 

Connecting the dots is a challenge even for scientists who are actively involved in inflammation research. One afternoon, I visited Ramnik Xavier, the chair of gastroenterology at Massachusetts General Hospital and an expert in Crohn’s disease and ulcerative colitis. The bowel is inflamed in both conditions: ulcerative colitis affects the colon, whereas Crohn’s disease can affect any part of the digestive system. But the nature of inflammation varies almost from person to person and involves interactions among DNA, many kinds of gastrointestinal cells, and the peculiarities of the gut microbiome. “Lots of cells, lots of genes, lots of bugs,” Xavier said.

Xavier, a compact man with a laconic manner and thick black hair marked by streaks of gray, initially studied the role of specialized white blood cells, known as T-cells and B-cells, in defending the body against the development of colitis. Eventually, with Mark Daly, a geneticist at the Broad Institute, Xavier began to search for genes that predispose people to inflammatory bowel disease and for genes that might protect them against it. The two scientists, as part of an international consortium, have identified at least a hundred and sixty areas of DNA that are associated with an increased risk of inflammatory bowel disease; Xavier’s lab has zeroed in on about two dozen genes within these regions of DNA.
One of the frustrations of treating inflammation is that our weapons against it are so imprecise. Drugs like naproxen and ibuprofen are the equivalent of peashooters. At the other extreme, cannon-like steroids shut down the immune system, raising the risk of infection, eroding the bones, predisposing the patient to diabetes, and causing mood swings. Even the peashooters can cause collateral damage: aspirin may help to protect against colon cancer, heart attack, and stroke, but it also raises the risk of gastrointestinal bleeding. Ibuprofen, naproxen, and similar drugs were labelled by the F.D.A. as increasing the risk of heart attack and stroke in people who’ve never suffered either condition, and clinical trials failed to show that they prevent or ameliorate dementia. (Although these drugs reduce inflammation, they may also alter the lining of blood vessels and increase the risk of clots.) Statins lower the chance of a heart attack, but there is growing concern not only about the side effect of muscle pain but also about increasing the likelihood of diabetes. And the absolute benefits of these preventive medications is slight, measured in single digits.

In the lab at the Broad Institute, Xavier and his team were trying to discover new treatments that can block inflammation in a targeted manner. The day I visited, they were assessing molecules associated with colitis, especially one called interleukin-10, or IL-10, which is known to decrease inflammation. In a cavernous room, I watched as a robotic arm moved among racks of plastic plates, each containing hundreds of small wells in which chemical compounds were being tested. Some people with Crohn’s disease have genetic mutations that disable the salubrious effects of IL-10. Xavier is trying to identify molecules that can compensate for this deficiency, in the hope that such molecules might eventually be turned into drugs to treat this subset of patients.

But other patients suffer from a different manifestation of Crohn’s—they can’t fully clear debris from cells in their gut, so it builds up, triggering inflammation. In a neighboring lab, members of Xavier’s research team were trying to develop drugs for that condition, too. A robotic arm was handling plates that contained genetically engineered cells and moving them under a fluorescent microscope. The images appeared on a computer screen—fields of cells studded with yellow and green dots, like the sky in van Gogh’s “Starry Night.”

On another visit, Xavier took me to his clinic at Mass General. Patients, ranging from the very young to the elderly, were reclining in Barcaloungers as nurses and physicians intravenously administered potent anti-inflammatory drugs. Later, I spoke by phone to one of Xavier’s patients, a forty-nine-year-old woman named Maria Ray, who received a diagnosis of colitis in 1998. She was treated with sulfa drugs and corticosteroids, which controlled the problem for several years, but in 2004, after a series of flare-ups, she underwent surgery to remove her colon. Soon after, she developed ulcers on her skin, arthritis of her knees and elbows, and inflammation in both eyes. Xavier prescribed other drugs, and for two years her condition improved, but lately her skin lesions and eye inflammation have returned. “We hoped surgery would cure her ulcerative colitis,” Xavier said. “But we don’t really understand why there is such an overactive immune system now inflaming these other parts of her body.”
At the very least, the fact that Ray has symptoms in many organs, despite the removal of her colon, complicates the simplistic view that treating the gut will suppress inflammation elsewhere. Moreover, there’s no evidence that patients with Crohn’s or colitis are more likely than average to develop dementia and other cognitive disorders. “What we see in mice is not always reproduced in people,” Xavier said.

 

Perhaps no aspect of inflammation is more compelling, or illusory, than the idea that it may be responsible for aging. An internist friend in Manhattan told me that healthy patients occasionally come in to her office carrying Mark Hyman’s books, eager to live longer by following his anti-inflammation life style. When I asked Hyman if he could introduce me to someone who follows his longevity regimen, he readily offered himself. “I’m aiming to live to a hundred and twenty,” he said.

The notion stems from grains of evidence, such as studies that have shown an increase in inflammation with age. The genesis of aging is still a mystery. It may occur for a host of reasons—a waning of the energy generated by the mitochondria within cells, the tendency of DNA to grow fragile and more mutation-prone over time—and it’s much too simplistic to attribute the process to inflammation alone. Luigi Ferrucci, the scientific director of the National Institute on Aging, conducted some of the early research on inflammation and aging, and for a while, he told me, he believed the avenue held promise. On the morning we spoke, he had just finished his daily six-mile run. Sixty-one years old, born in Livorno, on the coast of Tuscany, Ferrucci is an animated man with a stubbly beard who favors crew-neck sweaters. In the past four decades, he has studied thousands of people in order to identify the biological processes that result in aging. He measured scores of molecules in the blood, hoping to find clues that would lead him to the cause of aging’s hallmarks, particularly sarcopenia, or loss of muscle mass, and cognitive decline.

His most illuminating studies involved people in late middle age who showed no sign of heart disease, diabetes, dementia, or other conditions that might be associated with inflammation. He found that a single inflammatory molecule, called interleukin-6, was the most powerful predictor of who would eventually become disabled. Healthy patients with high levels of the IL-6 molecule aged more quickly and grew sicker than those without the inflammatory molecule. “I thought I had discovered the cause of aging and was going to win the Nobel Prize,” Ferrucci said, laughing.
But then he found other subjects with no evidence of inflammation, and without elevated levels of IL-6 or other inflammatory molecules, whose bodies nevertheless began to decline. “We are looking at the layer, not at the core of the problem,” he said. “Inflammation may accelerate aging in some people—but it is a manifestation of something that is occurring underneath.” He reiterated the point that correlation is not causation. “If you have the curiosity of the scientist, you can’t stop there, because you want to know why,” he said. “You want to break the toy so you can see how it’s working inside.”

Toward that end, Ferrucci recently organized a large team of collaborators and launched a new clinical study, GESTALT, which stands for Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing. Groups of healthy people will be studied intensively as they age, with detailed analyses of their DNA, RNA, proteins, metabolic capacity, and other sophisticated parameters, every two years for at least a decade. “Then we can say what mechanisms account for increased inflammation with aging, and the loss of muscle mass, or the loss of memory, or the loss of energy capacity or fitness,” Ferrucci said. “These have never really been addressed on a deep level in humans.”

In the meantime, he sticks to a Mediterranean diet, mainly out of fealty to his heritage. (Ferrucci is known among his N.I.H. colleagues as a gourmet Italian cook.) The media recently gave much attention to a study, published in 2013 in the New England Journal of Medicine, on the benefits of a Mediterranean diet in preventing heart attack or stroke. But, as Ferrucci noted, the benefits weren’t clearly related to inflammation and they accrued to a very small percentage of the subjects on the diet. “Believe me, if there were a diet that prevented aging, I would be on it,” he said.

We’d all like a simple solution for complex medical problems. We’re desperate to feel in command of our lives, particularly as we age and see friends and family afflicted by Alzheimer’s, stroke, and heart failure. “My patients, understandably, are very focussed on the foods they eat, wanting control, hoping they won’t have to take immune-suppressive treatments,” Gary Wu, the University of Pennsylvania gastroenterologist, told me.

Some years ago, I became obsessed with a restrictive diet—no bread, cheese, ice cream, cookies—in an attempt to lower my cholesterol levels. (My father died of a heart attack in his fifties, and I was haunted by his fate.) After nearly six months, I’d lost some fifteen pounds, but my cholesterol level had hardly budged, and I’d become so vigilant about everything I ate that I stopped enjoying meals. Gradually, I resumed a balanced and more reasonable diet and regained an appreciation for one of life’s fundamental pleasures.

Scientists may yet discover that inflammation contributes to disease in unexpected ways. But it’s important to remember, too, that inflammation serves a vital role in the body. “We are playing with one of the primary mechanisms selected by nature to maintain the integrity of our body against the thousand environmental attacks that we receive every day,” Ferrucci said. “Inflammation is part of our maintenance and repair system. Without it, we can’t heal.”

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Cholesterol and Regulation of Liver Synthetic Pathways

Curator: Larry H. Bernstein, MD, FCAP

 

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
http://dx.doi.org:/10.1172/JCI15593
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).

Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

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SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.

When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).

Figure 2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

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Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (916). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.

Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

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Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

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Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

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We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

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The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

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The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (2224). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

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In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.

Acknowledgments

Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.

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